I. Enhancer Sequences and Enhancer Binding PROTEINS
The development of higher eukaryotes is characterized by the differential, and tissue-specific, expression of certain genes. Among the factors that control such tissue-specific expression are diffusible positive transcriptional factors that are themselves produced in a tissue-specific manner. Certain of these transcriptional factors increase gene transcription by binding to specific DNA sequences known as "enhancer sequences" or enhancers" (Descombes, P. et al., Genes Devel. 4:1541-1551 (1990); see also Ptashne, M, Nature 335:683-689 (1988)).
Enhancer sequences are thus transcriptional regulatory sequences found in the DNA of animals and animal viruses (Khoury, G. et al., Cell 33:313-314 (1983); Schaffner, W. et al., Trends Genet. 1:224-230 (1985)). Enhancers are similar to promoters in that they act in cis, to regulate the transcription of gene sequences that are located on the same DNA molecule. They differ from promoter sequences in that their function does not depend on the position of the enhancer relative to the gene whose transcription is being enhanced. Enhancers can mediate transcription in either direction, and over considerable distances (up to several thousand base pairs).
The transcriptional factors that bind to enhancer sequences are termed "enhancer binding proteins" ("EBPs"). The ability of an enhancer to mediate gene expression in a particular cell is dependent upon the expression of an appropriate EBP in that cell. Thus, each enhancer is capable of mediating transcriptional enhancement either preferentially or exclusively in only those tissue types that express an EBP capable of binding to it.
CCAAT/enhancer binding proteins ("C/EBP") comprise a class of EBPs whose members are capable of preferentially recognizing and binding a CCAAT sequence motif (such as is found in the transferrin and ApoB genes), an enhancer core sequence motif, or the enhancer regions of several viral promoters (Landschultz, W. H. et al., Genes Dev. 2:786-800 (1989); Brunel, F. et al., J. Biol. Chem. 263:10180-10185 (1988); Metzger, S. et al., J. Biol. Chem. 265:9978-9983 (1990)).
The significance of human C/EBP has been elucidated by investigations of the rat and murine C/EBP analogs. The rat analog of C/EBP was initially isolated as a heat-stable protein from hepatic cells (Graves, B. et al., Cell 44:565-576 (1986); Johnson, P. F. et al., Genes Dev. 1:133-146 (1987)). High levels of expression of C/EBP were subsequently found to be limited to terminally differentiated cell types, such as adipocytes and hepatocytes, that play a central role in energy metabolism, particularly in the synthesis and mobilization of glycogen and fat (Christy, R. J. et al., Genes Dev. 3:1323-1335 (1989), Friedman, A. D. et al., Genes Dev. 3:1314-1322 (1989), McNight, S. L. et al., Genes Dev. 3:2021-2024 (1989)). In the adult mouse, for example, C/EBP is most abundant in the parenchymal cells of the liver and in adipose tissue. It has accordingly been proposed that C/EBP may modulate the transcription of genes that are expressed in tissues where synthesis or metabolism of lipids is an important part of their physiology (Birkenmeier, E. H. Genes Devel. 3:1146-1156 (1989)).
cDNA molecules encoding the rat and mouse analogs of C/EBP have been cloned (Landschultz, Genes Devel. 2:786-800 (1988); Xanthopoulos, K. G. et al., Proc. Natl. Acad. Sci. (U.S.A.) 86:4117-4121 (1989)), and expressed in tissue culture cells (Friedman, A. D. et al., Genes Devel. 3:1314-1322 (1989)). The predicted protein structures of the C/EBP analogs of the two species exhibit substantial homology. The C/EBP is present in single copy in the mouse genome, and is free of intervening sequences (Birkenmeier, E. H. Genes Devel. 3:1146-1156 (1989)).
The rat C/EBP analog is 42 kD, and is composed of 359 amino acids (Johnson, P. F. et al., Genes Dev. 1:133-146 (1987); Landschultz, W. H. et al., Genes Devel. 2:786-800 (1988)). The DNA binding domain is localized within the carboxy-terminal 80 residues (Friedman, A. D. et al., Genes Devel. 3:1314-1322 (1989)).
As indicated, C/EBPs comprise a family of related molecules that include, in addition to the C/EBP characterized by Landschultz, W. H. et al. (Genes Devel. 2:786-800 (1988)), "D-Binding Protein" (DBP) (Mueller C. R. et al., Cell 61:279-291 (1990)), and the "liver-enriched transcriptional activator protein ("LAP") (Descombes, P. et al., Genes Devel. 4:1541-1551 (1990). Additional members of the C/EBP family--designated CRP1, CRP2, and CRP3--were isolated by screening mouse and rat genomic libraries with DNA encoding a binding domain of C/EBP (Williams, S. C. et al., Genes Devel. 5:1553-1567 (1991).
Although significant progress has been made in identifying members of the C/EBP family, the use of these molecules, or the gene sequences that encode them, in the treatment of disease has not been reported. It would be desirable to identify therapeutic uses for these molecules and to develop means for targeting C/EBP molecules to cells and tissue in need of such therapies. The present invention provides such uses and targeting means.